Optical microchip array biosensor for multiplexed detection of bio-hazardous agents

An optical waveguide array biosensor suitable for rapid detection of multiple bio-hazardous agents is presented. SpectroSens? optical microchip sensors contain multiple spatially-separated waveguide channels with integral high-precision Bragg gratings sensitive to changes in refractive-index; selective surface-functionalisation of discrete sensing channels with different antibodies as bio-recognition elements enables selective multi-analyte biological detection. Interactions between target antigens in the test sample and respective surface-immobilised antibodies result in localised changes in refractive-index; the biosensor response manifests as increases in wavelength of light reflected from specific sensing channels. Multiplexed, label-free detection of 8 different biological agents, encompassing bacterial spores, vegetative cells, viruses and proteinaceous toxins has been demonstrated in real-time. Selective detection of Bacillus atrophaeus (BG) spores, Escherichia coli cells, MS2 viruses and ovalbumin (OVA) protein (simulant bio-hazardous agents) was first demonstrated as proof-of-concept; subsequently, detection of Bacillus anthracis (BA) spores (UM23CL2 strain), Franciscella tularensis (FT) cells (live vaccine strain), Vaccinia viruses (heat-killed) and ricin toxin (bio-hazardous agents) was proven. Two optical microchip sensors, each comprising 8 sensing channels were packaged into a single disposable cartridge allowing simultaneous 16-channel data acquisition. The specific antibody deposition sequence used in this study enabled detection of either 4 simulants or 4 bio-hazardous agents using a single consumable. The final device, a culmination of the multidisciplinary convergence of the fields of biology, chemistry, optoelectronics and microfluidics, is man-portable and inherently robust. The performance characteristics of the SpectroSens? technology platform highlight its potential for exploitation as a ‘detect to warn/treat’ biodetector in security and defence operations.     

Patterns of synchrony in natterjack toad breeding activity and reproductive success at local and regional scales

Empirical studies of the spatiotemporal dynamics of populations are required to better understand natural fluctuations in abundance and reproductive success, and to better target conservation and monitoring programmes. In particular, spatial synchrony in amphibian populations remains little studied. We used data from a comprehensive three year study of natterjack toad Bufo calamita populations breeding at 36 ponds to assess whether there was spatial synchrony in the toad breeding activity (start and length of breeding season, total number of egg strings) and reproductive success (premetamorphic survival and production of metamorphs). We defined a novel approach to assess the importance of short‐term synchrony at both local and regional scales. The approach employs similarity indices and quantifies the interaction between the temporal and spatial components of populations using mixed effects models. There was no synchrony in the toad breeding activity and reproductive success at the local scale, suggesting that populations function as individual clusters independent of each other. Regional synchrony was apparent in the commencement and duration of the breeding season and in the number of egg strings laid (indicative of female population size). Regional synchrony in both rainfall and temperature are likely to explain the patterns observed (e.g. Moran effect). There was no evidence supporting regional synchrony in reproductive success, most likely due to spatial variability in the environmental conditions at the breeding ponds, and to differences in local population fitness (e.g. fecundity). The small scale asynchronous dynamics and regional synchronous dynamics in the number of breeding females indicate that it is best to monitor several populations within a subset of regions. Importantly, variations in the toad breeding activity and reproductive success are not synchronous, and it is thus important to consider them both when assessing the conservation status of pond‐breeding amphibians.     

Idiosyncratic species effects confound size-based predictions of responses to climate change

Understanding and predicting the consequences of warming for complex ecosystems and indeed individual species remains a major ecological challenge. Here, we investigated the effect of increased seawater temperatures on the metabolic and consumption rates of five distinct marine species. The experimental species reflected different trophic positions within a typical benthic East Atlantic food web, and included a herbivorous gastropod, a scavenging decapod, a predatory echinoderm, a decapod and a benthic-feeding fish. We examined the metabolism–body mass and consumption–body mass scaling for each species, and assessed changes in their consumption efficiencies. Our results indicate that body mass and temperature effects on metabolism were inconsistent across species and that some species were unable to meet metabolic demand at higher temperatures, thus highlighting the vulnerability of individual species to warming. While body size explains a large proportion of the variation in species'' physiological responses to warming, it is clear that idiosyncratic species responses, irrespective of body size, complicate predictions of population and ecosystem level response to future scenarios of climate change.     

Complete nucleotide sequence of recD, the structural gene for the alpha subunit of Exonuclease V of Escherichia coli.

Intracellular amplification of the Escherichia coli RecB and RecC proteins does not result in an increase in Exonuclease V activity unless the level of a third protein, encoded between the recB and argA genes, is also amplified. Nucleotide sequence analysis of this region reveals a 1,824 nucleotide open reading frame which would encode a protein of 608 amino acids with a calculated molecular weight of 66,973. This is assumed to be the structural gene for the alpha subunit of Exonuclease V, recently designated recD. The proposed initiation codon of the recD gene overlaps the termination codon of the upstream recB gene by one nucleotide, suggesting that these genes may form an operon. The deduced amino acid sequence of the RecD protein contains a region which is homologous to highly conserved sequences in adenine nucleotide binding proteins.     

The effect of temperature on the growth,development and survival of Macropetasma africanus (Balss) (Penaeoidea:Penaeidae) larvae reared in the laboratory

Macropetasma africanus (Balss) has been successfully spawned and its larvae reared under controlled laboratory conditions. The relationship between egg number (E) and female total length (L) was E = 18.59 L^2.11. An experiment was designed to test the effect of temperature on larval development, survival and growth. Temperature effected larval development time, from 13–15 days at 25°C, to 25 days at 15°C (nauplius 1 to post-larva). Mortality was low for the naupliar stages at 25, 22 and 18°C, while at 15°C only 52% of the larvae reached nauplius 6. Mortality was highest from nauplius 6 to protozoea 1 (17, 21, and 18% at 25, 22, and 18°C, respectively), but decreased considerably for all temperatures once the mysis stage was reached. Overall survival rates from nauplius 1 to post-larva decreased with decreasing temperature (65, 54, 48, and 39% at 25, 22, 18, and 15°C respectively). Temperature also significantly affected larval growth. At 25°C mean total length was significantly (P < 0.05) larger than at 15°C (protozoea 2 to post-larva), while from protozoea 3 to post-larva total length differences were significantly different (P < 0.05) between 18 and 25°C. M. africanus has a major spawning peak in summer, suggesting that there may be a selective advantage to reproducing during the warmer months.     

Action of RecBCD enzyme on Holliday structures made by RecA

In vitro, Escherichia coli RecA protein acts upon gapped and partially homologous linear duplex DNA to generate recombination products linked by Holliday junctions. When strand exchange reactions are supplemented with purified RecBCD enzyme, we observe the formation of products that resemble "patch" recombinants. The formation of "splice" recombinant products was not observed. The individual subunits, RecB, RecC, or RecD, had no effect on RecA protein-mediated strand exchange nor on the Holliday junctions formed in the reaction. Analysis of the way in which patch products arise indicates exonucleolytic digestion of the linear arms of the recombination intermediates (alpha-structures) by RecBCD enzyme. We find no evidence for specific resolution events at the site of the Holliday junction by RecBCD enzyme using these DNA substrates.     

Hypoxanthine-guanine phosphoribosyltransferase deficiency: analysis of HPRT mutations by direct sequencing and allele-specific amplification

Summary The Lesch-Nyhan syndrome is a severe X chromosome-linked human disease caused by a virtual absence of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. A partial deficiency in the activity of this enzyme can result in gouty arthritis. To determine the genetic basis for reduction or loss of enzyme activity, we have amplified and sequenced the coding region of HPRT cDNA from four patients: one with LeschNyhan syndrome (HPRT_Perth) and three with partial deficiencies of HPRT activity, which have been designated HPRT_Urangan, HPRT_Swan and HPRT_Toowong. In all four patients, the only mutation identified was a single base substitution in exons 2 or 3 of the coding region, which in each case predicts a single amino acid substitution in the translated protein. Each base change was confirmed by allele-specific amplification of the patient's genomic DNA. It is interesting to note that the mutation found for HPRT_Perth is identical to that reported for HPRT_Flint. It appears that the two mutations are de novo events.     

Involvement of recB and recC genes of Escherichia coli in precise transposon excision

The mutations texA343 and texA344, which increase transposon excision, are complemented by multicopy plasmids carrying recC+ and recB+, respectively, but not by derivative plasmids in which the recC and recB genes are inactivated. This shows that texA343 and texA344 are mutations in recC and recB, respectively.     

Induction of protein synthesis in Escherichia coli following UV- or gamma-irradiation, mitomycin C treatment or tif Expression.

Summary The rate of synthesis of total cellular proteins has been studied by pulse labelling cells at various periods after irradiation with UV or -rays, after treatment with mitomycin C (MMC) or after expression of the temperature sensitive mutation tif. Subsequent gel electrophoresis and autoradiography reveals changes in the rate of synthesis of several proteins. The most striking change is in a protein of molecular weight 40,000, protein X, which has been previously most extensively studied in cells treated with nalidixic acid (Gudas, 1976). Synthesis of large quantities of protein X is induced by UV, -rays, MMC treatment or tif expression in rec ⁺ but not recA cells. A feature of recA cells is that they break down their DNA excessively after irradiation or MMC treatment. However, if protein synthesis following irradiation is prohibited by chloramphenicol, post-irradiation degradation becomes excessive in recA ⁺ cells. This inverse relationship between DNA degradation and new protein synthesis is consistent with the hypothesis that an induced protein such as X is responsible for controlling DNA degradation following irradiation. Protein X is not induced in a lexB mutant following MMC treatment. In this respect the lexB mutant behaves like lexA and recA mutants in that the ability to induce protein X can be correlated with excessive DNA degradation.Studies on the induction of proteins in inf, tif and tif sfi mutants fail to reveal any correlation between induction of protein X and either the induction of prophage or septation.